Lipid nanoparticles for wound healing

ABSTRACT

The present invention refers to lipid nanoparticles comprising a growth factor and/or an antimicrobial peptide and to the method for their preparation. Moreover, it refers to pharmaceutical compositions comprising such lipid nanoparticles, and a pharmaceutical acceptable carrier. Finally, it also refers to said pharmaceutical composition for its use as medicament and for its use in promoting wound healing, particularly by topical administration.

FIELD OF THE INVENTION

The invention refers to lipid nanoparticles and their use aspharmaceutical compositions for wound healing, in particular asnanoparticle suspensions, dressings or gels for topical application.

BACKGROUND OF THE INVENTION

Chronic wound treatment has become a major problem for health caresystems worldwide, representing a great economic and public healthchallenge. The current increase of risk factors such as, ageingpopulation, smoking, diabetes and obesity can complicate and slow downthe wound healing process, causing difficult-to-heal wounds.

Several palliative treatments including hyperbaric oxygen, negativepressure and surgical debridement are currently being used for thetreatment of chronic wounds in elderly people. In addition, a wide rangeof commercial synthetic dressings for the treatment of chronic ulcersare available in the market, although showing a limited healingefficacy.

The epidermal growth factor (EGF) plays an important role in tissueregeneration and repair by stimulating cell migration, differentiationand proliferation, and also by promoting granulation tissue formation.From a clinical point of view, EGF has been used to enhance woundhealing, especially in diabetic foot ulcers. Evidences of the beneficialeffects of topical EGF application in low-grade, neuropathic ulcers havebeen shown in clinical trials; however, the effect of topical EGFformulation can be abated, especially in high-grade wounds, since anincreased protease activity has been identified in this type of wounds.Rengen-D 150™ is a gel containing 150 μg/g of recombinant humanepidermal growth factor (rhEGF) manufactured in India for the treatmentof grade I or II diabetic ulcers. It requires twice dailyadministration, for an average treatment time of 6 weeks. Heberprot®, alyophilised formulation containing 75 μg of rhEGF, is administered threetimes weekly by intralesional injections. It is marketed in Algeria,Argentina, Colombia, Cuba, the Dominican Republic and Venezuela. A pilotstudy carried out in 20 diabetic patients demonstrated Herberprot® as afeasible and safe treatment to promote healing of chronic wounds inpatients with full thickness ulcers. However, rhEGF short half-liferequires continuous exposure (at least 6-12 hours) to enhance themitogenic effect on epithelial cells. Therefore, in order to achieve asignificant therapeutic effect in wound healing, it is necessary tooptimise the administration of growth factors such as rhEGF, in terms ofdose, delivery system and safety. For that purpose, Chu et al., 2010(Nanotechnology promotes the full-thickness diabetic wound healingeffect of recombinant human epidermal growth factor in diabetic rats”,Wound Repair Regen 2010; 15:499-505) have developed a method to preparenanoparticles of the polymer poly(lactic-co-glycolic acid) (PLGA) with a2% rhEGF content. rhEGF encapsulation efficiency was 85.6% and presenteda controlled release of biologically active rhEGF for up to 24 hours.Topical application once daily of these nanoparticles on full-thicknesswounds induced in diabetic rats promoted higher fibroblast proliferationlevels and fastest healing rates, compared with non-encapsulated rhEGF.

In addition, patent EP 1 987 817 B1 claims the process of producingpolymeric microspheres containing rhEGF for intralesional infiltrationinto the lower limbs of diabetic patients to prevent diabetic limbamputation. Developed microspheres with a 1.6-2.4% rhEGF content showeda rhEGF controlled release of 5 and 10 μg per day during 14 days and afaster injury healing in humans compared with equivalent amounts ofnon-encapsulated rhEGF.

Surprisingly, the inventors have developed lipid nanoparticlescomprising epidermal growth factor with high encapsulation efficiencies,that applied in a topical formulation twice a week, improve woundhealing.

BRIEF SUMMARY OF THE INVENTION

According to one aspect, the application refers to a lipid nanoparticle(nanoparticle of the invention) that comprises at least one solid lipidat room temperature, at least one non-ionic surfactant, and one growthfactor.

According to a further aspect, the application refers to a method forthe preparation of the lipid nanoparticle of the invention characterizedby comprising the following steps:

(i) preparing an aqueous solution comprising a non-ionic surfactant,(ii) preparing a lipophilic solution comprising a solid lipid at roomtemperature in an organic solvent,(iii) adding the aqueous solution (i) to the lipophilic solution (ii),subjecting the resulting mixture to sonication until obtaining anemulsion,(iv) evaporating the organic solvent of the obtained emulsion in (iii),and(v) collecting the lipid nanoparticles,wherein the growth factor is added to the solution (ii).

According to a further aspect, the application refers to apharmaceutical composition (pharmaceutical composition of the invention)comprising the lipid nanoparticle of the invention and a pharmaceuticalcarrier.

According to a further aspect, the application refers to the lipidnanoparticle of the invention and to the pharmaceutical compositioncomprising it, for its use as a medicament, and for its use in promotingwound healing.

Other objects, features, advantages and aspects of the presentapplication will become apparent to those skilled in the art from thefollowing description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graphic representation of the in vitro effect ofrhEGF-loaded lipid nanoparticles on cell proliferation.

FIG. 2 shows a micrograph of the cellular uptake of NileRed-SLN andNileRed-NLC formulations.

FIG. 3A shows a graphic representation of the in vivo effect ofrhEGF-loaded SLN, and

FIG. 3B shows a graphic representation of the in vivo effect ofrhEGF-loaded NLC on wound closure, and

FIG. 3C shows photographs of the wounds in untreated control rats andrats treated with empty SLN, empty NLC, rhEGF-MS, control empty MS, freerhEGF, rhEGF-SLN 20 μg, rhEGF-SLN 10 μg, rhEGF-NLC 20 μg, rhEGF-NLC 10μg. Data shown as means±standard deviation (S.D.) *Significantly greaterthan untreated control (*p<0.05, **p<0.01 and ***p<0.001);^(▪)Significantly greater than empty MS control (^(▪)p<0.05, ^(▪▪)p<0.01and ^(▪▪▪)p<0.001); ^()Significantly greater than empty SLN(^()p<0.05, ^()p<0.01 and ^()p<0.001); ^(▾)Significantly greaterthan empty NLC (^(▾)p<0.05, ^(▾▾)p<0.01 and ^(▾▾▾)p<0.001);^(∘)Significantly greater than free rhEGF (^(∘)p<0.05, ^(∘∘)p<0.01 and^(∘∘∘)p<0.001); ^(⋄)Significantly greater than rhEGF-MS 75 μg(^(⋄)p<0.05, ^(⋄⋄)p<0.01 and ^(⋄⋄⋄)p<0.001); ^(▴)Significantly greaterthan rhEGF-SLN 10 μg (^(▴)p<0.05, ^(▴▴)p<0.01 and ^(▴▴▴)p<0.001).

FIGS. 4A and 4C show a graphic representation of the in vitro effect ofrhEGF-loaded lipid nanoparticles on the inflammation score (A, C) and

FIGS. 4B and 4D show a graphic representation of the in vitro effect ofrhEGF-loaded lipid nanoparticles on the re-ephitelization score (B, D).

Data shown as means±S.D. *Significantly greater than untreated control(*p<0.05, **p<0.01 and ***p<0.001); ^(▪)Significantly greater thancontrol empty MS (^(▪)p<0.05, ^(▪▪)p<0.01 and ^(▪▪▪)p<0.001);^()Significantly greater than empty SLN (^()p<0.05, ^()p<0.01 and^()p<0.001); ^(▾)Significantly greater than empty NLC (^(▾)p<0.05,^(▾▾)p<0.01 and ^(▾▾▾)p<0.001); ^(∘)Significantly greater than freerhEGF (^(∘)p<0.05, ^(∘∘)p<0.01 and ^(∘∘∘)p<0.001); ^(⋄)Significantlygreater than rhEGF-MS 75 μg (^(⋄)p<0.05, ^(⋄⋄)p<0.01 and ^(⋄⋄⋄)p<0.001);^(▴)Significantly greater than rhEGF-SLN 10 μg (^(▴)p<0.05, ^(▴▴)p<0.01and ^(▴▴▴)p<0.001).

FIG. 5A shows micrographs (A) and FIG. 5B shows a graphic representation(B) of the coetaneous uptake of SLN-NileRed, NLC-NileRed andParaffin-NileRed (arbitrary pixel brightness values (ABU) corrected forthe background fluorescence in the stratum corneum, epidermis anddermis). *Significantly greater than Paraffin-NileRed (*p<0.05, **p<0.01and ***p<0.001). The inserted numbers give the respective enhancement ofpenetration over paraffin cream (PEE).

FIG. 6 shows a graphic representation of the in vitro effect of theLL37-loaded lipid nanoparticles on cell proliferation.

FIGS. 7A and 7B show a graphic representation of the in vitro effect ofthe combination of rhEGF and LL37 on cell proliferation.

FIG. 8A shows a graphic representation of the in vivo effect of emptyNLC, free rhEGF and rhEGF-NLC 20 μg on the wound closure in pigs on days15, 25, 36 and 43 and

FIG. 8B shows photographs of the wounds in pigs treated with empty NLC,free rhEGF and rhEGF-NLC 20 μg.

FIG. 9A shows a graphic representation of the epithelial length in thedifferent groups studied (empty NLC, free rhEGF and rhEGF-NLC 20 μg) ondays 15, 25, and 43 and

FIG. 9B shows histological images of the wounds on days 15, 25 and 43.Data shown as means±S.D. ***Significantly greater than empty NLC andfree rhEGF (p<0.001).

FIG. 10A shows a graphic representation of the wound healing extension(mm) on day 15, FIG. 10B shows a graphic representation of the woundhealing extension (mm) on day 25, and FIG. 10C shows a graphicrepresentation of the wound healing extension (mm) on day 43. Data shownas means±S.D. ***p<0.001 compared with the empty NLC and free rhEGFgroups.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention refers to a lipid nanoparticlecharacterized by comprising at least one solid lipid at roomtemperature, at least one non-ionic surfactant, and one growth factor.

It must be noted that as used in the present application, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise.

In the context of the present invention, lipid nanoparticles arereferred to particles in the nanometer range possessing a solid matrix.The matrix is composed of lipids being solid at room temperature, butalso at body temperature. In the case of solid lipid nanoparticles (SLN)the matrix consists of a solid lipid only. In case of nanostructuredlipid carriers (NLC) the matrix consists of a blend of solid lipids withliquid lipids (oils), but this blend being also solid at roomtemperature and also at body temperature (Muller et al., 2002, Solidlipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) incosmetic and dermatological preparations. Advanced Drug Delivery Reviews54 (1) S131-S155). The term “growth factor-loaded” refers to the growthfactor embedded or encapsulated in the matrix of the nanoparticle. Thus,in one particular embodiment, the lipid nanoparticles are SLN and inanother particular embodiment, the lipid nanoparticles are NLC. Thus, ina particular embodiment the lipid nanoparticles are SLN or NLC.

In a particular embodiment, the growth factor is selected from the groupconsisting of a growth factor belonging to the epidermal growth factor(EGF) family, transforming growth factor beta (TGF-beta) family,fibroblast growth factor (FGF) family, vascular endothelial growthfactor (VEGF), granulocyte macrophage colony stimulating factor(GM-CSF), platelet-derived growth factor (PDGF), connective tissuegrowth factor (CTGF), tumor necrosis factor-alpha family (TNFα),insulin-like growth factors (IGF). Preferably, the growth factor belongsto the epidermal growth factor (EGF) family, and more preferably thegrowth factor is the epidermal growth factor (EGF). In a particularembodiment, the lipid nanoparticles are EGF-loaded SLN (also referred asEGF-SLN hereinafter) and/or EGF-loaded NLC (also referred as EGF-NLChereinafter). In a particular embodiment of the invention, the epidermalgrowth factor is the recombinant human epidermal growth factor (rhEGF).Said rhEGF can be obtained commercially (Peprotech, Promega, Pharmchem,etc.) or produced by means of recombinant DNA technology, as describedfor example in Marioka-Fujimoto et al. (Modified enterotoxin signalsequences increase secretion level of the recombinant human epidermalgrowth factor in Escherichia coli. J Biol Chem. 1991 January 25; 266(3):1728-32), or in the patent application WO 91/18999 A1. In a particularembodiment, the lipid nanoparticle of the invention has a proportion ofgrowth factor, in particular EGF, comprised between 0.01% and 20% byweight with respect to the total weight of the lipid nanoparticle,preferably between 0.1% and 10% by weight, and more preferably between0.5% and 5% by weight with respect to the total weight of the lipidnanoparticle.

As mentioned above, the lipid nanoparticles of the present inventioncomprise at least one solid lipid at room temperature. In the context ofthe present invention, “solid lipid at room temperature” is understoodas the lipid being solid under 45° C., being able to be saturated orunsaturated. Said definition includes mono-, di-, or triglycerides,fatty acids, steroids and waxes. Likewise, derivatives of these fattyacids, understood as those compounds produced as a result of thereaction of the acid group with alcohols or amines such as, for example,the esters or amides of said fatty acids, can be used. Thus, in aparticular embodiment, the solid lipid at room temperature is selectedfrom acylglycerides, saturated fatty acids with a chain of at least 10carbon atoms or derivatives thereof and mixtures thereof. In a preferredembodiment the acylglycerides are selected from glyceryl palmitostearate(Precirol® AT05), glyceryl monostearate (Imwitor® 900) and glycerylbehenate (Compritol® 888ATO). In a more preferred embodiment, the lipidcomponent is glyceryl palmitostearate (Precirol® AT05).

In a particular embodiment of the invention, the lipid nanoparticle hasa proportion of solid lipid comprised between 1% and 40% by weight withrespect to the total weight of the lipid nanoparticle, preferablybetween 5% and 15.

As mentioned above, the lipid nanoparticles of the invention alsocomprise a non-ionic surfactant. The term “non-ionic surfactant” isunderstood as that compound that have a hydrophobic part and ahydrophilic part which allows producing an emulsion. In a particularembodiment of the present invention the non-ionic surfactant is selectedfrom polysorbates, polyethylene glycol copolymers and polypropyleneglycol copolymers, and mixtures thereof. In a particular embodiment, thenon-ionic surfactant is selected from the group consisting of Tween,Span, Poloxamer and mixtures thereof. In a preferred embodiment, thenon-ionic surfactant is Tween 80, and in another preferred embodiment,the non-ionic surfactant is a mixture of Tween 80 and Poloxamer.

In a particular embodiment of the invention, the proportion of non-ionicsurfactant is comprised between 0.01% and 10% by weight with respect tothe total weight of the lipid nanoparticle, preferably between 0.05% and5%.

In a particular embodiment of the invention, the lipid nanoparticle ofthe invention comprises between 1% and 40% of the solid lipid at roomtemperature, between 0.01% and 10% of the non-ionic surfactant, andbetween 0.01% and 20% of the growth factor, all percentages given byweight with respect to the total weight of the lipid nanoparticle.

In a particular embodiment, the lipid nanoparticle of the invention canoptionally be presented as lyophilized or desiccated product.

In a second aspect, the present invention refers to the lipidnanoparticles of the first aspect further comprising a liquid lipid atroom temperature. In the context of the present invention, “liquid lipidat room temperature” is understood as that lipid being liquid at roomtemperature and under 45° C., being able to be saturated or unsaturated.The liquid lipid at room temperature is selected from unsaturated orsaturated fatty acid esters, oils, fatty acids and triglycerides havinga chain with less than 10 carbon atoms, and their mixtures (for example,triglyceride of caprylic acid and capric acid (Miglyol®), soybean oil,isopropyl myristate, castoroil). In a preferred embodiment, Mygliol® isused as the liquid lipid. In a preferred embodiment, the lipid componentof the lipid nanoparticles is a combination of glyceyl palmitostearate(Precirol® AT05) and triglyceride of caprylic acid and capric acid(Miglyol®). In another variant of this second aspect of the invention,when a liquid lipid at room temperature is incorporated into thenanoparticle, it is in a proportion comprised between 1 and 30% byweight with respect to the total weight of the lipid nanoparticle,preferably between 5 and 15%. In another embodiment, the proportionsolid lipid:liquid lipid is between 0.5:10 and 5:10.

In a third aspect of the invention, the present invention refers to amethod (method 1 of the invention) for the preparation of the lipidnanoparticle of the first aspect of the invention defined in theparagraphs above, characterized by comprising the following steps:

(i) preparing an aqueous solution comprising a non-ionic surfactant,(ii) preparing a lipophilic solution comprising a solid lipid at roomtemperature in an organic solvent,(iii) adding the aqueous solution (i) to the lipophilic solution (ii),subjecting the resulting mixture to sonication until obtaining anemulsion,(iv) evaporating the organic solvent of the obtained emulsion in (iii),and(v) collecting the nanoparticles,wherein the growth factor is added to the solution (ii).

The lipid nanoparticles obtained with this method 1 are SLN. The firststep (i) consists of dissolving the non-ionic surfactant in an aqueoussolution, preferably water. The second step (ii) of preparing thelipophilic solution is carried out by means of dissolving the solidlipid at room temperature in an organic solvent, wherein the proportionof the solid lipid is at least 1% by weight with respect to the totalweight of the organic solvent. The growth factor, preferably EGF, andmore preferably rhEGF, is dissolved together with the lipid in theorganic solvent. The choice of the organic solvent depends in a largeextent on the lipid component. In a particular embodiment of theinvention, the organic solvent is selected from dichloromethane,acetone, chloroform and mixtures thereof, it is more preferablydichloromethane.

Once both solutions are prepared, the aqueous solution (i) is added tothe lipophilic solution (ii). The resulting mixture is then subjected tosonication until obtaining an emulsion. Subsequently, the organicsolvent is evaporated by means of any method known by a person skilledin the art. In a particular embodiment, the organic solvent evaporationstep is carried out by keeping the emulsion under mechanical stirringfor at least 60 minutes, preferably at least 120 minutes. After removingthe organic solvent, the lipophilic solution solidifies, and ananoparticle suspension is obtained, which is then filtrated bycentrifugation. Finally, collected lipid nanoparticles are washed andresuspended in purified water.

In a fourth aspect of the invention, the present invention refers to amethod (method 2 of the invention) for the preparation of the lipidnanoparticle of the second aspect of the invention defined in theparagraphs above, characterized by comprising the following steps:

(i) preparing an aqueous solution comprising a non-ionic surfactant,(ii) preparing a lipophilic solution comprising a blend of a solid lipidand a liquid lipid melted at a temperature higher than the melting pointof the liquid lipid,(iii) heat the aqueous solution (i) up to the same temperature than thelipophilic solution(iv) adding the aqueous solution (i) to the lipophilic solution (ii),subjecting the resulting mixture to sonication until obtaining anemulsion,(v) cooling down the emulsion (iv) at 5° C.±3° C. to allow lipidrecrystallization and nanoparticle formation, and(vi) collecting the nanoparticles,wherein a growth factor is added to the solution (ii).

The lipid nanoparticles obtained with this method 2 are NLC. The firststep (i) consists of dissolving the non-ionic surfactant in an aqueoussolution, preferably water. The second step (ii) of preparing thelipophilic solution is carried out by melting a blend of solid lipid anda liquid lipid at a temperature greater than the melting point of theliquid lipid. The growth factor, preferably EGF, and more preferablyrhEGF, is dissolved together with the blend of lipid.

Once the aqueous solution (i) is heated up to the temperature that theblend of lipids has been melted, the aqueous solution (i) is added tothe lipophilic solution (ii). The resulting mixture is then subjected tosonication until obtaining an emulsion. Subsequently, the emulsion (iv)is cooled down at a temperature of 5° C.±3° C. to allow lipidrecrystallization and nanoparticle formation. After lipidrecrystallization, a nanoparticle suspension is obtained, which is thenfiltered by centrifugation. Finally, collected lipid nanoparticles arewashed and resuspended in purified water.

A fifth aspect refers to the lipid nanoparticles obtained by the method1 and the lipid nanoparticles obtained by the method 2.

The lipid nanoparticles of the first, second and fifth aspects of thepresent invention are characterized by having a mean particle size equalto or less than 1 μm, they preferably have a mean size comprised between1 nm and 1000 nm, more preferably between 150 nm and 400 nm. The meansize can be measured by standard methods known by the person skilled inthe art, and which are described, for example, in the experimental partbelow (Table 1, Example 2). In addition, the lipid nanoparticles canhave a surface charge (measured by means of Z potential), the magnitudecan vary from −50 mV to +80 mV (Table 1, Example 2). Moreover, the lipidnanoparticles have an encapsulation efficiency greater than 40%, inparticular greater than 70% for SLN and greater than 95% for NLC (Table1, Example 2).

An important feature of the lipid nanoparticles of the present inventionis that they release the loaded growth factor in a sustained releasedmanner. Growth factor-loaded lipid nanoparticles present a releaseprofile characterized by an initial release (burst release) related tothe percentage of surface associated protein, followed by a fast releasephase from 4 hour to 24 hour and finally, a slower phase from 24 hoursto 72 hour is described ending with the release of the total amount ofgrowth factor (Table 3, Example 2).

This sustained release feature provides an important advantage since itenables safer treatments compared with those using free EGF, whichrequire continuous administrations of the growth factor and higherdosage to achieve the same therapeutic effect. In addition, by reducingthe dosage, further undesired adverse side effects can be decreasedsince administration of higher doses of the growth factor is no longernecessary because the encapsulated growth factor is released over thetime, in a smaller but more effective dose. Finally, sustained releaseof the growth factor allows decreasing the number of administrations,increasing patient treatment adherence and consequently, patient'squality of life.

It was surprisingly found by the inventors that the lipid nanoparticlesof the present invention, i.e. with EGF-loaded lipid nanoparticles, showan unexpected greater in vitro proliferation rate of fibroblasts thanfree EGF (Example 3, section 3.1, FIG. 1). Moreover, the lipidnanoparticles of the invention, i.e. EGF-SLN and EGF-NLC, are able toenter into the cell (Example 3, section 3.2, FIG. 2).

Interestingly, in vivo studies show that 4 topical administrations ofrhEGF-SLN in a dosage of 10 and 20 μg of rhEGF further improve woundhealing, in terms of wound closure (FIG. 3. A, C), inflammatoryresolution (FIG. 4.A) and re-epithelization process (FIG. 4.B) comparedwith 300 μg of rhEGF administered in 4 intralesional applications.Moreover, in vivo studies show that 4 topical administrations ofrhEGF-NLC in a dosage of 10 and 20 μg of rhEGF further improve woundhealing, in terms of wound closure (FIG. 3. B, C), inflammatoryresolution (FIG. 4.C) and re-epithelization process (FIG. 4.D) comparedwith 300 μg of rhEGF administered in 4 intralesional applications.Furthermore, obtained data reveal that the wound healing process washighly improved with 20 or 10 μg of rhEGF encapsulated in SLN and NLC,in terms of wound closure after 8 and 11 days of study, compared withone intralesional dose of 75 μg polylactic-co-glycolic acid (PLGA)microspheres rhEGF-MS. Thus, the use of lipid as a matrix material for ananoparticle is advantageous over the use of common biodegradable andhydrolytically degradable polymers, such as PLGA, because lipidnanoparticles allow topical administration of the molecule loaded in thelipid nanoparticles.

Additionally, the in vivo studies shown in the Example 6 (FIGS. 8-10)show that local and topical administration of rhEGF-NLC can enhancewound healing not only in terms of speed of wound healing and number ofhealed wounds (wound closure measurement), but also in terms of healingquality on the basis of the newly formed microvasculature, fibroblastmigration, collagen deposition and evolution of the inflammatoryresponse. In addition, these results are very relevant because theanimals treated with 20 μg rhEGF-NLC topically administeredsignificantly enhanced healing compared with those lesions treated with75 μg of free rhEGF intralesionally administered. These data, togetherwith the data from percentage of closed wounds and re-epithelization,demonstrate that rhEGF nanoencapsulation into rhEGF-NLC permits topicaladministration, and allows dose reduction because encapsulation preventsthe growth factor degradation in the wound site.

Interestingly, EGF-SLN and EGF-NLC show improved skin penetrationcapacity compared with paraffin cream (Example 3, section 3.4, FIG. 5).This is an important advantage because it allows the topicaladministration of the molecule loaded in the lipid nanoparticles.

Thus, a sixth aspect of the present invention refers to a pharmaceuticalcomposition comprising the lipid nanoparticle of the invention definedin the first, second and fifth aspect of the invention, as described inany of the paragraphs above, and a pharmaceutical carrier. In apreferred embodiment of this aspect, the pharmaceutical composition istopically administered. It can be administered in the form of a gel,cream, ointment, dressing or as a patch. Thus, in a particularembodiment, the pharmaceutical composition further comprises collagen,hyaluronic acid, aloe vera, fibrin, Carbopol® polymers and cellulosederivates.

Moreover, a seventh aspect of the present invention refers to the lipidnanoparticle defined in the first, second and fifth aspect of theinvention as described in any of the paragraphs above for its use as amedicament. Moreover, in an eight aspect, the invention refers to thepharmaceutical composition defined in the sixth aspect for its use as amedicament.

Taking into account the in vivo results described above, another aspectof the invention, ninth aspect of the invention, refers to the lipidnanoparticle defined in the first, second and fifth aspect of theinvention described in any of the paragraphs above, for its use inpromoting wound healing in a subject. Preferably, it refers toEGF-loaded SLN for its use in promoting wound healing in a subject, andto EGF-loaded NLC for its use in promoting wound healing in a subject.Moreover, in a tenth aspect, the invention refers to the pharmaceuticalcomposition defined in the sixth aspect for its use in promoting woundhealing in a subject. The term “subject” as used herein refers to anyvertebrate animal, preferably a mammal, and more preferably a human. Inthe context of the present invention, wound refers to chronic wounds,difficult-to-heal wounds, ischemic wounds and burns. In a particularembodiment, the wound is selected from the group comprising chronicwounds, isquemic wounds, burns and combinations thereof. Preferredchronic wounds are selected from the group consisting of diabetic footulcers, pressure ulcers, vascular ulcers and mixtures thereof.

In the context of the present invention, chronic wounds are defined aswounds that have failed to proceed through an orderly and timelyreparative process to restore anatomic and functional integrity over aperiod of three months. All wound types have the potential to becomechronic and, as such, chronic wounds are traditionally dividedetiologically into three categories: pressure, diabetic and vascularulcers (venous and arterial ulcers). A pressure ulcer is defined as anarea of localised damage to the skin and/or underlying tissue, usuallyover a bony prominence, as a result of pressure or shear, and/or acombination of these. Diabetic foot ulcers are one of the most fearedcomplications of diabetes due to the high consequences on the patientquality of life, and appear as a result of various factors, such asmechanical changes in conformation of the bony architecture of the foot,peripheral neuropathy (damaged nerves) and peripheral vascular disease(block arteries), all of which occurring with higher frequency andintensity in the diabetic population. Vascular ulcers are usuallylocalised on lower limbs. The great majority of vascular ulcers arechronic or recurrent. They cause considerable morbidity among patientswith peripheral vascular disease. Arterial or ischemic wounds are causedby poor perfusion to the lower extremities. Ischemia limits the supplyof nutrients and oxygen, killing the tissues and causing in the area theformation of an open wound. Reduced blood flow to the wound siteseverely impairs the healing response, causing a chronic wound that canlead to gangrene, and thus, to amputation. Finally, difficult-to-healwounds are characterized by the chronic persistence of inflammatorycells, disordered synthesis and remodelling of the extracellular matrix,and lack of re-epithelialization; and burns are damage to the skincaused by the effect of heat, fire, radiation, sunlight, electricity orchemicals.

It was surprisingly found by the inventors that the combination of EGFand the cathelicidin antimicrobial peptide LL37, show an unexpectedgreater cell proliferation than free EGF and free LL37, respectively(Example 4, FIG. 7). The LL37 peptide corresponds to the C-terminalfragment of the human cathelicidin anti-microbial protein, hCAP18, whichis a component of the innate immune system and has broad anti-microbialactivity (Heilborn et al., 2003, The cathelicidin antimicrobial peptideLL37 is involved in re-epithelization of human skin wounds and islacking in chronic ulcer epithelium. J Invest Dermatol 120(3): 379-389).Thus, a eleventh aspect of the present invention refers to a composition(composition of the invention) comprising lipid nanoparticles accordingto the first, second and fifth aspect of the present invention and lipidnanoparticles comprising at least one solid lipid at room temperature,at least one non-ionic surfactant and the LL37 peptide. In a particularembodiment of this aspect, the composition comprises EGF-loaded NLC andLL37-loaded NLC. In another particular embodiment, the compositioncomprises EGF-loaded SLN and LL37-loaded SLN. In another particularembodiment of this aspect, in the composition of the invention the ratioof growth factor-loaded, preferably EGF-loaded, lipid nanoparticles andLL37-loaded lipid nanoparticles is between 1:17 and 1:34. In anotherparticular embodiment, the composition comprises 15 ng/ml of EGF-loadedlipid nanoparticles and 0.25 to 5 μg/ml of LL37-loaded lipidnanoparticles.

A twelfth aspect of the present invention is a pharmaceuticalcomposition comprising the composition according to the eleventh aspectof the invention described in the last paragraph, and a pharmaceuticalcarrier. In a preferred embodiment of this aspect, the pharmaceuticalcomposition is topically administered. Thus, in a particular embodiment,the pharmaceutical composition further comprises collagen, hyaluronicacid, aloe vera, fibrin, Carbopol® polymers and cellulose derivates.

Furthermore, a thirteenth aspect of the present invention refers to thepharmaceutical composition of the twelfth aspect, for its use as amedicament.

A fourteenth aspect of the present invention, refers to thepharmaceutical composition defined in the twelfth aspect, for its use inpromoting wound healing in a subject.

A fifteenth aspect of the present invention, refers to a lipidnanoparticle comprising at least one solid lipid at room temperature, atleast one non-ionic surfactant and the LL37 peptide. The particularembodiments described in the first aspect of the present invention areapplicable to the LL37-loaded lipid nanoparticles of the fifteenthaspect, substituting the growth factor or EGF by the LL37 peptide.

A sixteenth aspect of the present invention, refers to the lipidnanoparticle of the fifteenth aspect further comprising a liquid lipidat room temperature. The particular embodiments described in the secondaspect of the present invention are applicable to the LL37-loaded lipidnanoparticles of this aspect substituting the growth factor or EGF bythe LL37 peptide.

A seventeenth aspect of the present invention, refers to a method(method 3 hereinafter) for the preparation of lipid nanoparticlesaccording to the fifteenth aspect of the present invention, which ischaracterized by the same steps of the method 1 described in the thirdaspect of the present invention, but wherein the LL37 peptide is addedto the lipophilic solution (ii), instead of the growth factor. Theparticular embodiments described for the method 1 described in the thirdaspect of the present invention are applicable to method 3, substitutingthe growth factor or EGF by the LL37 peptide.

An eighteenth aspect of the present invention, refers to a method(method 4 hereinafter) for the preparation of lipid nanoparticlesaccording to the sixteenth aspect of the present invention, which ischaracterized by the same steps of the method 2 described in the fourthaspect of the present invention, but wherein the LL37 peptide is addedto the lipophilic solution (ii), instead of the growth factor.

A nineteenth aspect of the present invention refers to the lipidnanoparticles obtainable by the method 3 and the lipid nanoparticlesobtainable by the method 4, described in aspects seventeenth andeighteenth, respectively.

Regarding LL37, this peptide can be synthesized using an automaticpeptide synthesizer and standard methods for peptide syntheses.Moreover, it can be obtained commercially, for example fromSigma-Aldrich. LL37 has the aminoacid sequence SEQ ID NO 1(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES).

An important feature of the LL37-loaded lipid nanoparticles of thepresent invention is that they release the loaded LL37 in a sustainedreleased manner. LL37-loaded lipid nanoparticles present a releaseprofile characterized by an initial release (burst release) related tothe percentage of surface associated peptide, followed by a fast releasephase from 4 hours to 24 hours and finally, a slower phase from 24 hoursto 72 hours is described ending with the release of the total amount ofLL37 (Table 4, Example 2).

Surprisingly, the inventors show that the LL37-loaded lipidnanoparticles, have an unexpected greater in vitro proliferation rate offibroblasts than free LL37 (Example 3, section 3.3, FIG. 6). Thus, atwentieth aspect of the present invention refers to the LL37-loadedlipid nanoparticles of the fifteenth, sixteenth and nineteenth aspectsof the present invention, for their use as a medicament.

A twenty first aspect of the present invention, refers to theLL37-loaded lipid nanoparticles of the fifteenth, sixteenth andnineteenth aspects of the present invention, for its use in promotingwound healing in a subject.

A twenty second aspect of the present invention refers to apharmaceutical composition comprising the LL37-loaded lipidnanoparticles of the fifteenth, sixteenth and nineteenth aspects of thepresent invention, and a pharmaceutical acceptable carried. In apreferred embodiment of this aspect, the pharmaceutical composition istopically administered. Thus, in a particular embodiment, thepharmaceutical composition further comprises collagen, hyaluronic acid,aloe vera, fibrin, Carbopol® polymers and cellulose derivates.

The present invention also refers, in a twenty third aspect, to thepharmaceutical composition of the twenty second aspect for its use as amedicament.

A twenty fourth aspect of the present invention refers to thepharmaceutical composition defined in the twenty second aspect, for itsuse in promoting wound healing

Finally, a twenty fifth aspect of the present invention refers to a kitcomprising any one of the lipid nanoparticles described in the first,second, fifth, fifteenth, sixteenth and nineteenth aspect of the presentinvention, or mixtures thereof. Additionally, it refers to a kitcomprising a pharmaceutical composition according to any of thepharmaceutical compositions described in the sixth, twelfth and twentysecond aspect of the invention.

The examples below serve to further illustrate the invention, and toprovide those of ordinary skill in the art with a complete disclosureand description of how the lipid nanoparticles herein are prepared andevaluated, and are not intended to limit the scope of the invention. Inthe examples, unless expressly stated otherwise, amounts and percentagesare by weight, temperature is in degree Celsius or is at ambienttemperature, and pressure is at or near atmospheric.

EXAMPLES Example 1 Lipid Nanoparticle Preparation

SLN and NLCs were prepared by the emulsification-ultrasonication basedmethod (Muller et al., 2002; Che et al., 2010 (Effects of lipophilicemulsifiers on the oral administration of lovastatin from nanostructuredlipid carriers: Physicochemical characterization and pharmacokinetics.European Journal of Pharmaceutics and Biopharmaceutics 74, 474-482).

In case of rhEGF-loaded SLN, 10 ml of 1% (v/v) Tween 80 aqueous solutionwas added to a 2 ml of a dicloromethane solution containing 0.1% (w/v)commercial rhEGF and 10% (w/w) Precirol® ATO 5. Immediately after, themixture was emulsified for 30 seconds at 50 W (Branson® 250 sonifier,CT, USA). This step produced an o/w emulsion which was then stirred for2 hours to extract the organic solvent and to obtain particle hardening.The SLN were then collected by centrifugation at 2500 rpm for 10 minutesusing a centrifugal filter unit with 100 kDa on pore size (Amicon®Ultra, Millipore, Spain), and washed three times with miliQ water.Finally, trehalose was added as cryoprotectant in an aqueous solution of15% respect to Precirol® ATO 5.

Concerning the preparation of rhEGF-loaded NLC, a warm aqueous solutionof 0.67% (w/v) Poloxamer and 1.33% (w/v) Tween 80, heated at 40° C. for1 minute, was poured into a 200 mg of melted Precirol® ATO 5 basedmixture containing 2 mg of commercial rhEGF and 20 mg of Miglyol, alsoheated at 40° C. for 1 minute. The resulting blend was then emulsifiedfor 15 seconds at 50 W (Branson® 250 sonifier, CT, USA) and stored for12 hours at 4° C., recrystallising the lipid, to allow NLC formation.Finally, particles were collected, washed and lyophilised as previouslydescribed. The target loading of rhEGF both in SLN and NLC was 1% (w/w).

The LL37-loaded SLN and LL37-loaded NLC were prepared as described forthe rhEGF-loaded SLN and rhEGF-loaded NLC, respectively, but usingsynthetic (Sigma-Aldrich 94261) or recombinant LL37 peptide, instead ofrhEGF.

Example 2 Lipid Nanoparticle Characterization

2.1.—The mean size (z-average) and polydispersity index (PI) weremeasured by photon correlation spectroscopy. Each assay was performed intriplicate before and after nanoparticle lyophilization. Zeta potential(ζ) was determinated through Doppler velocimetry (LDV). All themeasurements described above were assessed using a Malvern® Zetasizer3000 instrument (Malvern Instruments, Worcestershire, UK). Surfaceappearance and sphere morphology was determined through scanningelectron microscopy (SEM; Jeol® JSM-35 CF) and transmission electronmicroscopy (TEM).2.2.—The encapsulation efficiency (EE) was calculated indirectlymeasuring free rhEGF and free LL37 (non-encapsulated) removed by thefiltration/centrifugation technique used to collect the SLN and NLC.Each sample was diluted 1:10000 with Dulbecco's phosphate bufferedsaline (DPBS) solution containing 0.05% (v/v), Tween 20 and 0.1% (w/v)Bovine Serum Albumin (BSA). The amount of free rhEGF and free LL37 wasestimated using a commercially available Sandwich Enzyme-LinkedImmunosorbent assay kit for human EGF (human EGF ELISA development kit,Peprotech) and for human LL37 (Human LL-37 ELISA development kit, Hycultbiotech), following manufacturer's instructions. Encapsulationefficiency (EE) was assessed applying the following equation:

${E\; E\mspace{14mu} (\%)} = {\frac{{{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {rhEGF}\mspace{14mu} \left( {{LL}\; 37} \right)} - {{Free}\mspace{14mu} {rhEGF}\mspace{11mu} \left( {{LL}\; 37} \right)}}{{Total}\mspace{14mu} {amount}\mspace{14mu} {of}\mspace{14mu} {rhEGF}\mspace{14mu} \left( {{LL}\; 37} \right)} \times 100}$

All tests were performed in triplicate, and results are reported as themeans±S.D. of these assays.

TABLE 1 rhEGF-loaded SLN and NLC characterization: Before liophylizationAfter liophylization Zeta potential Formulation Size (nm) PDI Size (nm)PDI (mV) EE (%) SLN 226.1 ± 0.8  0.28 310.9 ± 15.8 0.30 −33.8 ± 0.3 73.9± 2.2 NLC 241.5 ± 23.3 0.38 353.6 ± 4.53 0.37 −34.6 ± 0.2 95.7 ± 4.7

TABLE 2 LL37-loaded SLN and NLC characterization: Before liophylizationAfter liophylization Zeta potential Formulation Size (nm) PDI Size (nm)PDI (mV) EE (%) SLN 251.53 ± 7.79 0.35 ± 0.02 340.27 ± 2.42 0.25 ± 0.01−33.97 ± 0.57 70.67 ± 0.22 NLC  225.4 ± 1.26 0.35 ± 0.07 320.90 ± 3.720.37 ± 0.01 −33.47 ± 1.78 97.52 ± 0.75

As shown in Tables 1 and 2, the PDI values were less than 0.5 in all theformulations before and after freeze-drying, confirming the homogeneoussize distribution of the formulations in all cases. Reconstitution ofdry particles for size measurements did not present any inconvenience,suggesting the absence of particle fusion or aggregation, despite theincrease in size. Regarding to the zeta potential, both formulationspresented similar surface charge, of approximately −34 mV. In addition,ELISA assays demonstrated that NLC EE was slightly higher than SLN EE.

In both formulations the surface of nanoparticles was smooth and porefree. In contrast, both SEM images and TEM images suggested that SLNwere more regular in size than NLC (data not shown).

2.3.—In Vitro Release Studies

The release study was conducted by incubating 32 mg and 23 mg of SLN orNLC (corresponding to −200 μg of rhEGF or LL-37) in 2 ml of 0.02 Mphosphate-buffered saline (PBS) for three days. At selected intervals,the release medium was removed by filtration/centrifugation and replacedby the same quantity of PBS. The amount of rhEGF and LL37 was assayed byELISA using the protocol described in section 2.2.

TABLE 3 Cumulative percentage of rhEGF released over time rhEGFcumulative rhEGF cumulative Time release from SLN release from NLC 30minutes 25.15 ± 3.19  26.88 ± 3.19  4 hours 38.50 ± 10.09 44.79 ± 5.02 8 hours 57.11 ± 13.44 61.31 ± 5.04 24 hours 71.20 ± 17.36 75.87 ± 5.8848 hours 88.27 ± 23.80 86.10 ± 6.23 72 hours 102.24 ± 24.59  98.95 ±8.02

TABLE 4 Cumulative percentage of LL37 released over time LL37 cumulativeLL37 cumulative Time release from SLN release from NLC 30 minutes 27.60± 2.96  31.21 ± 10.56  4 hours 45.19 ± 6.82 52.14 ± 4.57  8 hours 55.87± 4.31 65.18 ± 6.21 24 hours  62.51 ± 21.22  74.93 ± 10.20 48 hours 89.00 ± 12.41 93.81 ± 5.25 72 hours 100.76 ± 19.20 105.70 ± 10.25

The in vitro rhEGF and LL37 release profiles displayed in Table 3 andTable 4, respectively, show that both formulations presented a similarrelease behaviour. Firstly an initial release (burst release) related tothe percentage of surface associated protein or peptide, followed by afast release phase from 4 hours to 24 hours and finally, a slower phasefrom 24 hours to 72 hours is described ending with the release of thetotal amount of rhEGF and LL37.

Example 3 Cell Proliferation, Cellular Uptake and Skin Penetration ofNanoparticles

3.1.—Effect of rhEGF-Loaded Nanoparticles on Cell Proliferation

24-well plate was used for the proliferation assay. 35000 Balb/C 3T3fibroblast resuspended in 1 ml of completed culture medium (DMEMsupplemented with 10% FCS) were seeded in each well. After 8 hours ofincubation, the medium was replaced by 1 ml of 0.2% FCS supplementedDMEM and cells were incubated overnight. Then, medium was replaced by 1ml of: (i) 0.2% FCS-supplemented DMEM, (ii) 15 ng/ml of free rhEGF in0.2% FCS-supplemented DMEM, (iii) empty SLN in 0.2% FCS-supplementedDMEM, (iv) 15 ng/ml of rhEGF-loaded SLN (rhEGF-SLN) in 0.2%FCS-supplemented DMEM, (v) empty NLC in 0.2% FCS-supplemented DMEM and(vi) 15 ng/ml of rhEGF-loaded NLC (rhEGF-NLC) in 0.2% FCS-supplementedDMEM.

The cells were cultured under the same conditions for 24 hours, 48 hoursand 72 hours. The experiments were performed in triplicate. After thedifferent incubation intervals, 100 μl of CCK-8 (Sigma-Aldrich, SaintLouise, USA) was added to each well (Zhou, Y., Qian, M., Liang, Y., Liu,Y., Yang, X., Jiang, T., Wang, Y., 2011. Effects of Leukemia InhibitoryFactor on Proliferation and Odontoblastic Differentiation of HumanDental Pulp Cells. J. Endod. 37, 819-824). After 4 hours of incubation,absorbance was read at 450 nm and at 650 nm as the reference wavelength.The absorbance was directly proportional to the number of living cellsin culture.

The proliferation assay carried out in Balb/c 3T3 cells demonstrated themitogenic effect of rhEGF. FIG. 1 shows greater cell proliferation ingroups treated with rhEFG (rhEGF-NLC, rhEGF-SLN and free rhEGF) thancontrol groups, after 48 hours and 72 hours. Strikingly, rhEGFencapsulated into lipid nanoparticles (either SLN or NLC) showed agreater increase in the mitogenic effect of Balb/C 3T3 fibroblast thanfree rhEGF.

3.2.—The Effect of LL37-Loaded Nanoparticles on Cell Proliferation

The experiment was carried out as described in section 3.1 but withHUVEC cells (Human Umbilical Vein Endothelial Cells). The tested groupswere the following: (i) 0.2% FCS-supplemented DMEM, (ii) 0.5 and 0.1μg/ml of free LL37 in 0.2% FCS-supplemented DMEM, (iii) empty SLN in0.2% FCS-supplemented DMEM, (iv) 0.5 and 0.1 μg/ml of LL37-loaded SLN(LL37-SLN) in 0.2% FCS-supplemented DMEM, (v) empty NLC in 0.2%FCS-supplemented DMEM, (vi) 0.5 and 0.1 μg/ml of LL37-loaded NLC(LL37-NLC) in 0.2% FCS-supplemented DMEM and (vii) 10% DMSO as negativecontrol.

The proliferation assay carried out on HUVEC cells demonstrated themitogenic effect of LL37. FIG. 6 shows greater cell proliferation ingroups treated with LL37 (LL37-NLC, LL37-SLN and free LL37) than controlgroups for both doses after 48 hours.

3.3.—Cellular Uptake of NileRed-SLN and NileRed—NLC Formulations

50000 Balb/C 3T3 fibroblast and 100000 HaCaT keratynocites were culturedseparately on coverslips in complete culture medium for 24 hours. Themedium was then replaced with 1 ml of assay medium. The following groupswere tested: (i) 25 μg of NileRed-SLN in completed DMEM and (ii) 25 μgof NileRed-NLC in completed DMEM. After 1 hour incubation, cells werewashed and fixed. Nuclei were then stained with DAPI (500 ng/ml) andcoverslips were mounted into the slides for examination in afluorescence microscope.

The results obtained from the cellular uptake study are presented inFIG. 2. The cell cytoplasm turns red (white arrows in FIG. 2) due to theinternalization of the SLN and NLC. The figure shows the capability ofthe SLN-NileRed and NLC-NileRed to enter into the cell. No differenceswere observed between the uptake capability of SLN and NLC.

3.4.—Coetaneous Uptake of SLN and NLC Formulations

The experiment was carried out as described by Küchler et al., 2009(Nanoparticles for skin penetration enhancement—A comparison ofdendritic core-multishell-nanotransporter and solid lipid nanoparticles.Eur J Pharm Biopharm.; 71:243-250). The skin of the same animal was usedfor treated and control experiments. The groups assayed were (n=3) (i)SLN-NileRed, (ii) NLC-NileRed and (iii) Nile red loaded paraffin creamused as a reference. All preparations had Nile red at identicalconcentration of 0.004%. Skin was maintained for 24 hours at 32° C. Skinwas then washed with PBS, dried and cut into vertical slices (bottom tosurface) of 20 μm thickness using a freeze microtome Frigocut 2800 N,Leica, Bensheim, Germany. Slices were stored at −20° C. and analysedwithin 24 hours, subjecting then to normal light and fluorescence light.

For all experiments, the corrected arbitrary pixel brightness values ABUobtained from dye uptake were related to uptake data from Nile redloaded paraffin cream. The resulting parameter called penetrationenhancing effect (PEE) was calculated for all skin layers. The value ofthe paraffin cream was 1 by definition (Lombardi Borgia, S., Regehly,M., Sivaramakrishnan, R., Mehnert, W., Korting, H. C., Danker, K.,Roder, B., Kramer, K. D., Schafer-Korting, M., 2005. Lipid nanoparticlesfor skin penetration enhancement-correlation to drug localization withinthe particle matrix as determined by fluorescence and parelectricspectroscopy. J. Control. Release 110, 151-163).

Penetration enhancing effect (PEE) values, estimated from the correctedarbitrary pixel brightness (ABU) values of the skin sections arepresented in FIG. 5. PEE values showed that dye penetration ofSLN-NileRed and NLC-NileRed exceed those found with the paraffin creamin all the skin slides. PEE values for SLN-NileRed and NLC-NileRedincreased fourfold in the stratum corneum (4.74 and 4.62 respectively)and about twofold in epidermis (2.09 and 2.03 respectively). A minor,yet still often significant enhanced penetration was also observed inthe dermis (1.29 and 1.32 respectively). In short, the ABU and PEEvalues over 1 demonstrate an improved skin penetration capacity of SLNand NLC compared with the paraffin cream. Furthermore, data also reflectthe capability of these nanoparticles to deliver Nile Red to the skinand therefore rhEGF and LL37.

Example 4 In Vitro Proliferation of rhEGF/LL37/Combination rhEGF andLL37

The experiment was carried out as described in section 3.1. The synergyeffect of LL37 and rhEGF was tested in Balb/C fibroblast and HaCatkeratynocites. 24-well plate was used for the proliferation assay. 35000Balb/C 3T3 fibroblast resuspended in 1 ml of completed culture medium(DMEM supplemented with 10% FCS) were seeded in each well. After 8 hoursof incubation, the medium was replaced by 1 ml of 0.2% FCS supplementedDMEM and cells were incubated overnight. Medium was then replaced by 1ml of: (i) 0.2% FCS-supplemented DMEM, (ii) 15 ng/ml of free rhEGF,(iii) 5, 0.5 and 0.25 μg/ml of LL37, (iv) 15 ng/ml of rhEGF and 5, 0.5and 0.25 μg/ml of LL37. Cells were cultured at 37° C. and 5% CO₂atmosphere for 24 hours, 48 hours and 72 hours.

The proliferation assay carried out in HaCaT (human queratinocytes) andBalb/C fibroblast demonstrated the after 48 hours, 10 and 50 ng/ml LL37and 15 ng/ml rhEGF higher enhanced mitosis than free rhEGF alone (FIG.7).

Example 5 In Vivo Wound Healing with rhEGF-Loaded SLN and rhEGF-LoadedNLC

In vivo wound healing efficacy of rhEGF-loaded SLN and rhEGF-loaded NLCwas tested against two different rhEGF formulations. (i) 75 μg oflyophilised rhEGF (similar to commercialised Heberprot®) and (ii) 75 μgof rhEGF encapsulated in 1% (w/w) loaded polymeric microspheres(rhEGF-MS 75 μg) prepared in our laboratory by the combination withalginate and polylactic-co-glycolic acid (PLGA) using double-emulsionmethod as described in the European patent application EP12382476.Briefly, 2 ml of dichloromethane:acetone (3:1) solution containing 5%(w/v) of PLGA (Resomer RG503) was emulsified by sonication for 15seconds at 50 W with 0.2 ml of an internal aqueous phase (in miliQwater) containing 0.05% (w/v) rhEGF, 2.5% mg (w/v) of Human SerumAlbumin (HSA), 0.25% (w/v) of polyethylene glycol 400 (PEG400) and 2.5%(w/v) sodium alginate MVG (Pronova UP, NovaMatrix FMC BioPolymer,Sandvika, Norway). The resulting emulsion (w₁/o) was poured into 15 mlaqueous solution containing 5% polyvinyl alcohol (PVA) and 5% NaCl, andemulsified using a paddle stirrer during 60 seconds to obtain the doubleemulsion (w₁/o/w₂). Finally, 400 ml of an aqueous solution of 5% NaCland 0.6 mM of calcium chloride was added and stirred for 30 minutes. Themicrospheres were then collected by filtration and lyophilized.

5.1.—Animals

Eighty 8-week-old male db/db mice were used. Genetically diabetic db/dbmice (BKS.Cg-m+/+ Lepr^(db)/J) were obtained from Javier Laboratories(Saint Berthevin Cedex). All procedures were performed according toprotocols approved by the Institutional Animal Care and Use Committee ofthe University of the Basque Country.

5.2.—Experimental Procedure

The experiments were performed by adapting the procedure used byMichaels et al.

(2007) (db/db mice exhibit severe wound-healing impairments comparedwith other murine diabetic strains in a silicone-splinted excisionalwound model. Wound Repair and Regeneration 15, 665-670). Animals weredivided into the following 10 groups (n=8): (i) untreated control, (ii)75 μg of free rhEGF, (iii) empty MS, (iv) empty SLN, (v) empty NLC, (vi)rhEGF-MS 75 μg, (vii) rhEGF-SLN 10 μg, (viii) rhEGF-SLN 20 μg (ix)rhEGF-NLC 10 μg and (x) rhEGF-NLC 20 μg.

Nanoparticles previously resuspended in 20 μl of vehicle (0.5%carboxymethylcellulose in 0.9% saline) were administered topically twicea week with a micropipette and were allowed to spread over the woundbed. Free rhEGF resuspended in 0.5 ml of vehicle was intralesionallyadministered twice a week by deepening the needle downward into thewound. rhEGF-MS 75 μg also resuspended in 0.5 ml of vehicle wasintralesionally administered once in the day of wound induction.

5.3.—Evaluation of Wound Healing

The effectiveness of treatments in improving wound healing was evaluatedby measuring the wound area (cm²) on the day of surgery and on days 4,8, 11 and 15 after wound induction (FIG. 3B), using a digital camera(Lumix FS16, Panasonic®, Spain) and an image analysis program (ImageJ®,Biophotonics Facility, University of McMaster, Canada). The woundclosure was expressed as the percentage area of the initial wound size.

All experimental groups (rhEGF-SLN 20 and 10 μg, rhEGF-NLC 20 and 10 μgand rhEGF-MS 75 μg) presented greater wound area reduction from day 4(FIG. 3A) compared with their control groups (for rhEGF-SLN groups theircontrols are free rhEGF, empty SLN and untreated control; for rhEGF-NLCgroups their controls are free rhEGF, empty NLC and untreated control;and for rhEGF-MS 75 μg its controls are free rhEGF, empty MS anduntreated control). No statistical differences were found between bothlipid nanosparticle formulations (rhEGF-SLN and rhEGF-NLC), in contrastwound contraction became significantly greater than rhEGF-MS 75 μg andfree rhEGF (p<0.001). Regarding control groups, rhEGF-MS 75 μg alsoshowed statistically significant wound closure. By day 8, woundcontraction reached the greatest difference among all experimentalgroups and their controls. Again, no differences were found amongrhEGF-SLN, rhEGF-NLC and rhEGF-MS 75 μg formulations. Interestingly, 11days after wounding, the animals treated with rhEGF-SLN 20 μg showedsignificantly further wound reduction than the other experimental groups(p<0.05 for rhEGF-SLN 10 pg, rhEGF-NLC 20 and 10 μg and p<0.001 forrhEGF-MS 75 μg) and their control groups. However, the otherexperimental groups also presented wound contraction albeit milder. Atthe end of the study, by day 15 wounds from the groups treated with allthe lipid nanoparticle formulations had almost closed. Surprisingly,rhEGF-MS 75 μg presented retarded wound contraction (73.16±3.24%)compared with lipid nanoparticles (˜95%). It is also remarkably thatmultiple free rhEGF administrations showed fewer differences among allthe control groups than those presented by the nanoformulated rh-EGFgroups.

TABLE 5 Wound healing Experimental groups 4^(th) day (%) 7^(th) day (%)11^(th) day (%) 14^(th) day (%) Untreated Control 5.04 ± 5.71 9.86 ±7.94 46.76 ± 3.77 61.12 ± 2.85 Empty MS 7.35 ± 4.36 9.63 ± 5.85 36.94 ±6.46 59.55 ± 4.82 Empty SLN 2.66 ± 5.25 19.50 ± 8.07  42.18 ± 8.82 59.53± 5.37 Empty NLC 3.57 ± 7.95 13.51 ± 11.43  35.14 ± 11.54 48.38 ± 7.57Free rhEGF 8.42 ± 3.96 19.94 ± 5.21  44.80 ± 2.64 61.83 ± 2.99 rhEGF-MS75 μg 22.10 ± 7.73  43.12 ± 3.97  62.31 ± 4.88 73.16 ± 3.24 rhEGF-SLN 10μg 17.03 ± 7.61  42.70 ± 14.40  71.05 ± 18.53 92.33 ± 9.05 rhEGF-SLN 20μg 21.08 ± 16.29 45.02 ± 13.84 88.40 ± 3.53 97.75 ± 1.67 rhEGF-NLC 10 μg17.88 ± 10.55 44.28 ± 9.16  68.80 ± 8.15 93.64 ± 3.95 rhEGF-NLC 20 μg24.64 ± 10.17 43.81 ± 6.20  70.10 ± 8.47 90.11 ± 6.17

5.4.—Histological Estimation of Wound Healing

On days 8 and 15, animals were killed through CO₂ inhalation. The woundsand surrounding tissue (˜1 cm) were excised and fixed in 3.7%paraformaldehyde for 24 hours. The fixed tissues were then bisected,embedded in paraffin and excised in 5-μm thick layers. The samples wereprocessed by H&E staining for morphological observations.

Regarding the resolution phases of inflammatory recovery (FIG. 4.A) andwound maturity, the scale described by Cotran et al. (2000) was used(Cotran, R., Kumar, V., Collins, T., 2000. Reparación de los tejidos:regeneración celular y fibrosis. Patologia estructural y funcional: McGraw Hill Interamericana, 2000, pp 95-120). The score of each wound wasdetermined in a semi-quantitative within a range from 0 to 4. 0: Absenceof inflammatory response. 1: acute inflammation (formation of the fibrinclot and pyogenic membrane; migration of leucocytes and polynuclearneutrophils), 2: predominance of diffuse acute inflammation(predominance of granulation tissue and pyogenic membrane; vascularneogenesis), 3: predominance of chronic inflammation (fibroblastproliferation), 4: resolution and healing (reduction or disappearance ofchronic inflammation although occasional round cells may persist).

The re-epithelization process (FIG. 4.B, D) was measured according tothe criteria established by Sinha et al. (Sinha, U.K., Gallagher, L. A.,2003. Effects of steel scalpel, ultrasonic scalpel, CO2 laser, andmonopolar and bipolar electrosurgery on wound healing in guinea pig oralmucosa. Laryngoscope 113, 228-236). 0: re-epithelization at the edge ofthe wound, 1: re-epithelization covering less than half of the wound, 2:re-epithelization covering more than half of the wound, 3:re-epithelization covering the entire wound with irregular thickness and4: re-epithelization covering the entire wound, normal thickness.

5.5.—Statistical Analysis

All data are expressed as the means±standard deviation. Based on theLevene test result of homogeneity of variances, the means were comparedthrough student's t test or one-way ANOVA for multiple comparisons.Subsecuently, the Bonferroni or Tamhane post-hoc test was applied.Differences were considered significant at p<0.05. Computations wereperformed using SPSS 20.0 (SPSS®, Inc., Chicago, Ill.).

By day 8, only rhEGF-SLN 20 μg presented a chronic inflammatory stateclose to the complete resolution, in which fibroblast proliferationprevailed (score 3.67±1.15) (Table 6). Animals treated with rhEGF-NLC 20μg, almost with predominance of acute inflammatory state near to thechronic state, showed less inflammatory score (2.75±0.46). However,these differences were not statistically significant. In addition, bothformulations presented significantly greater restoration than theircontrol groups. Conversely, rhEGF-SLN 10 μg and rhEGF-NLC 10 μg did notleave the acute inflammatory state (<2.00); nevertheless, differences(p<0.001) were found among these groups and the untreated control group,which showed absence of inflammatory response. rhEGF-NLC 10 μg alsopresented differences (p<0.05) with its control (empty NLC).

Regarding the evaluation of dose response (20 or 10 μg) between thelipid nanoparticles, only rhEGF-SLN 20 μg presented an improvedresolution (higher than rhEGF-SLN 10 μg).

Histopathological analysis also indicated that in wounds treated withrhEGF prevailed a diffuse acute inflammatory state, with an inflammatoryscore of 2.13±0.64 (significantly greater than their control groups).These differences did not achieve the statistical significance with anyof the lipid nanosphere (neither rhEGF-SLN nor rhEGF-NLC).

As mentioned above, 15 days after wounding differences tend to be lower,not reaching the statistical significance among any group and theircontrols.

Regarding the re-epithelization grade, by day 8 obtained data showedthat only the groups treated with lipid nanoparticles with the highestdose (20 μg) and animals treated with rhEGF-MS 75 μg, presented newepithelium covering more than half of the wound (>2.00, related to theSinha U.K. (2003) criteria). Moreover, statistically significantdifferences were found among these groups and their control groups (freerhEGF, empty SLN and untreated control for rhEGF-SLN 20 μg and freerhEGF, empty NLC and untreated control for rhEGF-NLC 20 μg) (FIG. 5). Incontrast, in groups treated with lipid nanoparticles with the lower doseof rhEGF (rhEGF-SLN 10 μg and rhEGF-NLC 10 μg), the new epithelium didnot overtake more than half of the wound. Furthermore, althoughsignificant differences were found with the untreated group (p<0.01),the statistical significance tended to loose with empty SLN in the caseof rhEGF-SLN 10 μg, and with empty NLC in the case of rhEGF-NLC 10 μg,even though the re-epithelized scores were higher for the 10 μgrhEGF-loaded lipid nanoparticles (FIG. 4.B). Regarding rhEGF-MS 75 μg,by day 8 the re-epithelized area covered more than half of the wound andstatistically significant differences were found among rhEGF-MS 75 μgand its control groups (untreated control and empty MS).

Thus, the study of re-epithelization process (Table 7) revealed thatanimals treated with 4 doses of 75 μg of free rhEGF showed higherre-epithelization score than untreated groups (1.25±0.89 and 0.00±0.00respectively). Surprisingly, the effect of multiple intralesional dosesof free rhEGF showed less re-epithelization improvement than thatobtained from 4 topical administrations of rhEGF-SLN and rhEGF-NLC and 1intralesional dose of rhEGF-MS 75 μg.

15 days after wound induction the re-epithelization study did not revealdifferences among groups.

TABLE 6 Inflammation score Inflammation score Day 8 Day 16 Untreatedcontrol 0.13 ± 0.51 1.83 ± 0.98 Empty MS 0.25 ± 0.46 2.83 ± 1.33 EmptySLN 1.25 ± 0.46 2.88 ± 0.83 Empty NLC 1.00 ± 1.10 2.50 ± 1.38 Free rhEGF1.75 ± 0.71 2.75 ± 0.95 rhEGF-MS 75 μg 2.13 ± 0.64 2.86 ± 0.90 rhEGF-SLN10 μg 1.63 ± 1.51 3.50 ± 0.93 rhEGF-SLN 20 μg 3.67 ± 1.15 3.38 ± 0.52rhEGF-NLC 10 μg 1.88 ± 1.21 3.13 ± 0.35 rhEGF-NLC 20 μg  2.75 ± 0.46*,3.00 ± 0.00

TABLE 7 Re-epithelization score Re-epithelization score Day 8 Day 16Untreated control 0.00 ± 0.00 2.50 ± 1.76 Empty MS 0.38 ± 0.52 3.00 ±1.10 Empty SLN 1.00 ± 0.00 3.38 ± 0.92 Empty NLC 1.00 ± 0.89 3.00 ± 0.89Free rhEGF 1.25 ± 0.89 2.71 ± 0.49 rhEGF-MS 75 μg 2.13 ± 0.64 3.17 ±0.98 rhEGF-SLN 10 μg 1.71 ± 0.76 3.00 ± 0.93 rhEGF-SLN 20 μg 2.33 ± 0.583.25 ± 0.71 rhEGF-NLC 10 μg 1.50 ± 1.05 3.38 ± 0.52 rhEGF-NLC 20 μg 2.75± 0.74 32.86 ± 0.69 

Example 6 In Vivo Wound Healing in Pigs with rhEGF-Loaded NLC6.1.—Animals

All the protocols and procedures used were previously approved by theInstitutional Animal Care and Use Committee from the Jesús UsónMinimally Invasive Surgery Centre (JUMISC). Six female Large White pigs,with a mean weight of 26.82±2.90 kg at the beginning of the study,distributed in individual pens of 2.90 m×1.35 m were used. All theanimals of the study were randomly distributed in acclimated rooms,where the following housing conditions were established: 12-hourlight-dark cycle, temperature between 20 and 25° C., eight air changesper hour with HEPA-filtered ventilation and relative humidity between 50and 70%. As pen environmental enrichment, hanging chains and chewingrubber toys were installed as chewable elements. After anacclimatization period, the animals were identified by ear tag codes tobe included in the study.

6.2.—Wound Model and Surgical Procedure

After 12 hours of solid and 6 hours of liquid starvation, the animalswere sedated through intramuscular administration of 15 mg/kg ketamineand 0.2 mg/kg diazepam. Anaesthesia was then induced by propofol (3mg/kg) administration to allow endo-tracheal intubation. Immediatelyafter, the animals were connected to an anaesthetic machine through acircular circuit attached to a ventilator supplying sevoflurane asanaesthetic agent at a concentration of 2.7% in an oxygen flow of 1L/min. As analgesia, remifentanil was used through intravenous infusionin a continuous infusion rate of 0.1 μg/kg/min during the surgery.

To each animal 6 wounds (6 cm×5 cm) were created leaving a minimum of1.5-2.0 cm between each ulcer, after tattooing the ulcer edges using aframe to have permanent reference of the initial wound area. Ulcers werecreated using monopolar diathermy in coagulation mode to obtain ischemicwound edges, 2 mm in depth, leaving the panniculus adiposus.Postoperative analgesia was administered throughbuprenorphine-containing transdermal patches and systemic antibiotictreatment with amoxicillin/clavulanic acid (20 mg/kg) during one week.

6.3.—Experimental Groups

Treatment administration began 24 hours after wound induction (day 1 ofthe study) to allow primary homeostasis, platelet adhesion andaggregation, and activation of the coagulation cascade. The animals wererandomly divided into three groups (n=2): (i) empty NLC, (ii) 20 μgrhEGF-NLC and (iii) 75 μg free rhEGF. Nanoparticles previouslyresuspended in 150 μl of vehicle (0.5% carboxymethylcellulose in 0.9%saline) were topically administered twice a week by spreading them overthe wound bed. Free rhEGF, resuspended in 1 ml of vehicle, wasintralesionally administered twice a week.

Throughout the study, the wounds were covered to avoid dehydration andmicrobial contamination. Moreover, dressing prevented scab formation andfacilitated observation of the epithelized edges for area measurementsof the healed wounds. To this end, each wound was covered with aparaffin gauze to avoid bandage adhesion, on top three sterile cottongauzes were placed, fixed with an adhesive bandage and a stickingplaster. Dressings were changed twice per week to continually assess thestate and evolution of the wound, maintaining this way a high asepticlevel. The cleaning process was carry out with the greatest care, usingsterile gauzes and saline solution to eliminate exudates and detritus,respecting the integrity of the newly formed granulation tissue.

6.4.—Blood Samples

To monitor the general health status of the animals, blood samples werecollected to check the haematological and biochemical levels on the dayof the wound induction, on day 15 and at the end of the study (day 43).The studied parameters were: haematocrit, haemoglobin, mean corpuscularvolume (MCV), mean corpuscular haemoglobin (MCH), white blood cells,total proteins and platelets.

In addition, plasma samples from the rhEGF-NLC and free rhEGF treatedanimals were collected to assess rhEGF systemic absorption. rhEGFdetection was performed by ELISA (human EGF ELISA development kit,PeproTech). Samples were drawn when the plasma rhEGF levels wereexpected to be higher. Thus, samples from the animals treated withrhEGF-NLC were collected on day 1 of the experiment (prior to thetreatment administration), 4 hours and 24 hours after administration.Plasma samples from the animals treated with free rhEGF were obtainedjust after administration and 30 minutes after the first dose.

6.5.—Serial Wound Analysis of Healed Wounds

Wound healing kinetic was determined by measuring the wound closure(percentage of initial wound area closed) on days 1, 15, 25, 36 and 43.The wound area was assessed, in a standardized manner, taken photographsperpendicularly to the wound surface, using the same illumination andplacing a transparent plastic sterile ruler next to the wounds tointroduce a metric reference in the pictures for further processing. Thewound area was calculated using the image software ImageJ (see section5.3). The wounds were considered healed when closure was above 95%.

6.6.—Histological Assessment of Wound Healing

Complete skin thickness biopsies from the centre of the unhealed woundto the healthy margin (2 mm) were collected using a sterile scalpel.Collected samples were immediately fixed in 4% formalin, embedded inparaffin and excised in 5-μm thick layers. Samples were stained withhaematoxilyn-eosin (HE) for morphological observations. Biopsies werecollected on day 15 for wounds 1, 2 and 3 and on days 25 and 43 for allof the wounds. On day 36, wound images were taken but no biopsies werecollected not to delay wound healing.

Wound healing was determined in terms of the re-epithelization grade bymeasuring the newly formed epithelia, and wound maturity and healingquality according to the Cotran et al. (2000) criteria.

6.7.—Haematology Analysis

The haematology analysis did not reveal any change in none of thestudied parameters throughout the study. In addition, all the obtainedvalues were within the normal ranges for healthy pigs (data not shown).

Plasma samples of the animals treated with rhEGF-NLC and free rhEGF werecollected to assess rhEGF absorption into the systemic circulation whenthe rhEGF concentration was expected to be higher in plasma, because thesystemic use of rhEGF has been limited by the concern of abnormalepithelial growth. The plasma collection time points were chosen basedon the EGF half-life, the administration route and the delayed releaseof the growth factor loaded in the rhEGF-NLC. In this regard, becausethe rhEGF incorporated into rhEGF-NLC was expected to be absorbed laterthan free rhEGF due to encapsulation, plasma sampling was performed onday 1 of the experiment, 4 hours and 24 hours after rhEGF-NLCadministration, instead of 30 minutes after administration, as performedwith the animals treated with free rhEGF.

Due to human and pig marked similarities, similar rhEGF plasmaconcentrations were expected. As illustrated in Table 8, the rhEGFplasma concentration showed values of approximately 0, clearly bellowbasal human EGF concentration (0.4 ng/ml). This fact is very strikingbecause the virtually nonexistence of systemic absorption, even whenfree rhEGF is directly injected into the wound, may minimize the siteeffects; and therefore, improve treatment safety and ensure rhEGF localeffect in the lesion.

TABLE 8 rhEGF plasma concentrations. The data are shown as the means ±S.D. rhEGF plasma concentrations (ng/ml) 30 minutes 4 hours 24 hoursrhEGF-NLC — 0.03 ± 0.01 0.01 ± 0.01 Free rhEGF 0.20 ± 0.01 — —

6.8.—Serial Wound Analysis of Healed Wounds

Enhanced healing was evaluated by calculating the number of healedwounds in each experimental group on days 15, 25, 36 and 43. Thegreatest differences among the groups were found on day 25 of theexperiment. As depicted in FIG. 8, by day 15 none of the wounds hadcompletely closed. By day 25 the percentage of healed wounds weresignificantly higher for the rhEGF-NLC treated group than for the emptyNLC treated group. In addition, it should be pointed out that treatmentwith rhEGF-NLC showed a slight superior effectiveness compared with freerhEGF (50% and 40%, respectively). Although the percentage of healedwounds were similar in both groups, these data are particularlysignificant as the wounds treated with rhEGF-NLC received two weeklytopical administrations of 20 μg of rhEGF, while those wounds treatedwith free rhEGF received higher doses (75 μg twice a week)intralesionally administered. By day 36, almost all the wounds hadhealed completely; the wounds treated with rhEGF (both with rhEGF-NLCand free rhEGF) were completely closed and the wounds treated with emptyNLC reached 90% healing. At the end of the study (day 43) all the woundswere completely healed.

6.9.—Histological Assessment of Wound Healing 6.9.1.—Re-EpithelizationGrade

As shown in FIG. 9A, by day 15 the length of the new epithelia wassignificantly greater in the animals treated with 20 μg rhEGF-NLCtopically administered than in those wounds treated with empty NLC andthose receiving 75 μg free rhEGF intralesionally administered (p<0.001).The improved re-epithelization effectiveness of rhEGF-NLC compared withfree rhEGF, suggests that nanoencapsulation protects the growth factoragainst the wound microenvironment and reduces the inactivation exertedby the proteases and oxidative stress of the wound area. This protectionmay be responsible for the enhanced effectiveness of rhEGF-NLC observedin the in vivo studies. However, the differences among the groups didnot reach the statistical significance on days 25 y 43 (FIG. 9A), eventhough the mean values obtained by day 25 for those animals treated withrhEGF (free or encapsulated) were higher than those for the empty NLCgroup.

6.9.2.—Wound Maturity and Healing Quality

Due to the extent of the created lesions, in the same histologicalsample different healing stages can be observed according to the Cotranet al. (2000) criteria. As depicted in FIG. 10A, by day 15 the animalstreated with rhEGF-NLC showed not only a statistically significant lowerextension of inflammatory tissue (Grade 2) compared with those woundsreceiving empty NLC and free rhEGF, but also a significantly higherextension of almost completely healed epidermis (Grade 4) (p<0.001). Incontrast, empty NLC and free rhEGF treated wounds mainly showed adiffuse acute inflammation with presence of some polymorphonuclearneutrophils, granulation tissue formation and vascular neogenesis (Grade2).

By day 25, because the healing process continued in all the studiedgroups, the extension of grade 2 was significantly smaller than thatobserved on day 15 and the extension of grade 3 and grade 4significantly higher (p<0.001). In addition, it is noteworthy that eventhough on day 25 differences in the re-epithelisation length among thegroups could not be found, differences were still appreciated in woundmaturity and healing quality (FIG. 9A and FIG. 10B). The animals treatedwith rhEGF-NLC showed a healing grade closer to complete healing thanthose groups receiving empty NLC and free rhEGF, which still showedchronic inflammation and a granulation tissue rich in new vessels andfibroblast proliferation.

In addition, as shown in FIG. 9B and FIG. 100, by day 43 all the woundsof all the experimental groups showed some chronic inflammation (grade 3and 4). However, lesions treated with rhEGF-NLC presented asignificantly improved healing grade and wound maturity (p<0.001).

1. Lipid nanoparticle characterized by comprising at least one solidlipid at room temperature, at least one non-ionic surfactant, and onegrowth factor.
 2. Lipid nanoparticle according to claim 1, where thegrowth factor is the epidermal growth factor.
 3. Lipid nanoparticleaccording to claim 2, where the epidermal growth factor is recombinanthuman epidermal growth factor (rhEGF).
 4. Lipid nanoparticle accordingto claim 1 further comprising at least a liquid lipid at roomtemperature.
 5. Lipid nanoparticle characterized by comprising at leastone solid lipid at room temperature, at least one non-ionic surfactant,and the cathelicidin antimicrobial peptide LL37.
 6. Lipid nanoparticleaccording to claim 5 further comprising at least a liquid lipid at roomtemperature.
 7. Composition characterized by comprising a lipidnanoparticle according to claim
 1. 8. A medicament comprising a lipidnanoparticle according to claim
 1. 9. A method of promoting woundhealing in a subject comprising using a lipid nanoparticle according toclaim
 1. 10. The method according to claim 9, wherein the wounds areselected from the group consisting of diabetic foot ulcers, pressureulcers, vascular ulcers, isquemic wounds and combinations thereof. 11.Pharmaceutical composition comprising a lipid nanoparticle according toclaim 1, and a pharmaceutically acceptable carrier.
 12. Pharmaceuticalcomposition comprising a composition according to claim
 7. 13.Pharmaceutical composition according to claim 11, for its use inpromoting wound healing in a subject.
 14. Kit comprising a lipidnanoparticle according to claim
 1. 15. Method for the preparation of thelipid nanoparticle according to claim 1, comprising the following steps:(i) preparing an aqueous solution comprising a non-ionic surfactant,(ii) preparing a lipophilic solution comprising a solid lipid at roomtemperature in an organic solvent, (iii) adding the aqueous solution (i)to the lipophilic solution (ii), subjecting the resulting mixture tosonication until obtaining an emulsion, (iv) evaporating the organicsolvent of the emulsion obtained in (iii), and (v) collecting thenanoparticles, wherein a growth factor or the LL37 peptide is added tothe solution (ii).
 16. Method for the preparation of the lipidnanoparticle according to claim 4, comprising the following steps: (i)preparing an aqueous solution comprising a non-ionic surfactant, (ii)preparing a lipophilic solution comprising a blend of solid lipids andliquid lipids melted at a temperature higher than the melting point ofthe liquid lipid, (iii) heat the aqueous solution (i) up to the sametemperature than the lipophilic solution (ii), (iv) adding the aqueoussolution (i) to the lipophilic solution (ii), subjecting the resultingmixture to sonication until obtaining an emulsion, (v) cooling down theemulsion (iv) at 5° C.±3° C. to allow lipid recrystallization andnanoparticle formation, and (vi) collecting the nanoparticles, wherein agrowth factor or LL37 peptide is added to the solution (ii).